Cancer is the cause of 1 in 4 deaths in the US and is the leading cause of disease-related death in children.1 Even with the most current standard of therapy including surgical removal of the tumor, chemotherapy, and irradiation therapy, pediatric and adolescent sarcomas remain a significant challenge with 1 in 3 patients developing relapsed or metastatic tumors that are incurable.2 One promising strategy to combat these aggressive tumors is the use of oncolytic herpes simplex viruses (oHSVs) that are genetically engineered to selectively replicate in cancer cells and destroy the tumors using the virus's natural replication cycle.4-6 However, patient response to oHSV therapy varies greatly in clinical trials, with some patients achieving dramatic cures while the majority of patients having no improvement with oHSV therapy.7 It is unclear what determines individual patient response, but there are likely microenvironment factors that differ between patients. It has been assumed in the research community that oncolytic virus efficacy is dependent on the direct lytic effect of virus replication in the tumor cells.4-6 However, our sarcoma models are discordant with this dogma between oHSV replication and anti-tumor efficacy. It is known that classically activated M1 macrophages produce reactive oxygen species that are cytotoxic to the tumor, while alternatively activated M2 macrophages secrete growth factors and immunosuppressive signals that promote tumor growth.8, 10, 15-17 However, the effect of M1/M2 macrophage subtypes on oncolytic virus efficacy against sarcoma tumors is unknown. The objective of this study is to test our hypothesis that M2 tumor associated macrophages (TAMs) diminish oHSV anti-tumor efficacy and modulation of macrophage polarization improves oHSV therapy. To determine if TAMs are responsible for sarcoma resistance against oHSV, sarcoma tumor bearing mice treated with or without macrophage depleting liposomal clodronate will be evaluated for oHSV anti-tumor efficacy by measuring tumor progression, virus replication, vasculature staining, and immune cell infiltration. Then the extent of TAM polarization on tumor resistance to oHSV by coculturing unstimulated M0 or cytokine activated M1 and M2 macrophages with sarcoma cells and measuring oHSV cytotoxicity, virus replication, cytokine secretion, and changes in macrophage polarization. Finally, trabectedin chemotherapeutic will be used to deplete M2 macrophages and test for improvement in oHSV anti-tumor efficacy will be evaluated as described above along with quantification of M1/M2 tumor infiltrating macrophage populations. Understanding the role of the individual M1/M2 macrophage subtypes in oHSV resistance will lead to refined therapies that selectively target the tumor associated macrophage subtype that causes oHSV resistance, our studies may also result in the identification of biomarkers that predict oHSV efficacy in individual cancer patients.